Advances in molecular cloning techniques have led to the identification and isolation of an expanding array of genes with mutations responsible for human diseases. Such advancements have made it possible to consider gene transfer, i.e., replacement of absent or mutated genes, as a potential treatment for certain genetic and/or acquired disorders.
Traditional gene therapy approaches utilize ex vivo gene transfer; Liebert et al., Human Gene Therapy, 2:251-256 (1991). Ex vivo approaches involve transformation of cells in vitro with DNA (the cells having first been harvested from the patient and grown in culture), followed by introduction of the transformed cells back into the patient. In such techniques, in vitro transfer is generally done using retrovirus-based vectors; Zwiebel et al., Science, 243:220-222 (1989). Ex vivo gene therapy looks quite promising; however, such therapy is obviously limited by the fact that it can only be used to transfect a limited number of cells and cannot be used to transfect cells which are not first removed from the body.
A theoretically more attractive gene therapy approach currently under investigation is direct gene transfer technology, i.e, direct introduction of a gene transfer vector into a target organ in vivo. This technique utilizes purified gene sequences as drug molecules in a manner similar to the way conventional pharmaceutical agents are administered. Currently, both viral-based and nonviral-based DNA transfer methods are being tested in vivo, with a variety of purified genes, and in a variety of clinical settings.
At present, direct gene transfer technology is being extensively studied in the treatment of pulmonary disorders. The lung is a particularly attractive organ for such therapy because it is directly accessible via the airways, is isolated from other organ systems, and its anatomy allows for different routes of administration to be contemplated. In vivo transfection of lung tissue after administration of genes to the lungs has been achieved by a variety of means; see e.g., Canonico et al., J. Appl. Physiol., 77:415-19 (1994); Stewart et al., Hum. Gene Ther., 3:267-75 (1992); Wilson et al., Hum. Gene Ther., 5:1019-57 (1994). Vector systems have included adenoviruses; see e.g., Brody et al., Ann N.Y. Acad. Sci., 716:90-101 (1994); Mastrangeli et al., J. Clin. Invest., 91:225-34 (1993), retroviruses; Han et al., Am. J. Respir. Cell Mol. Biol., 11:270-78 (1994), and liposome based complexes; Stewart et al., Hum. Gene Ther., 3:267-75 (1992) and references cited therein.
These demonstrations of transfection and production of protein illustrate the promise that in vivo gene therapy of the lung may have; however, there are still many crucial problems that need to be addressed before such therapy can prove to be a realizable goal. For example, viral approaches, while appearing to be the most efficient in terms of DNA transfer, are likely to be severely limited due to immunogenicity, especially where repeat dosing is expected. Furthermore, the fact that retroviruses can only transfect dividing cells and must integrate into the host cell chromosome for transcription to occur, brings into question the safety of in vivo use of such vectors, in particular, the possibility of recombination with endogenous viruses, which could in turn mutate into a deleterious infectious form. Finally, there are likely to be substantial formulation, scale up and manufacturing problems associated with use of virus-based vectors.
Non-viral techniques, although less likely to cause a host reaction, have so far demonstrated poor transfection efficiency in vivo. For example, techniques utilizing DNA-liposome complexes to transfer genes require large doses of DNA and lipid in order to detect even minute levels of protein after instillation in the lungs; Stribling et al., P.N.A.S. USA, 89:11277-81 (1992). Likewise, aerosol delivery to animals and humans is a highly inefficient process in that 80% to 90% of the starting material can be wasted irrespective of the inhalation device employed. These transfection efficiency problems must be overcome in order to make in vivo gene transfer using nonviral vectors clinically applicable. Resolution of the transfection efficiency problem requires that large quantities of inexpensive DNA plasmid be readily available and/or a formulation be found that dramatically improves protein production in vivo.